Cochlear outer hair cells (OHCs) are unique sensory-motor cells responsible for signal amplification and frequency discrimination in the mammalian inner ear. Strong evidence suggests that the motile response of OHCs to chemical or electrical stimulation provides the mechanical effectors to the "cochlear amplifier," the active mechanism that enhances approximately 100-fold the peak of the sound-induced basilar membrane motion. Our long-term goal is to understand the molecular mechanisms involved in the regulation of OHC motility. Based on previous studies in non-auditory cell populations, we hypothesize that OHC motility is regulated by structural and functional changes in the cytoskeleton. The recent identification of the small Rho GTPases RhoA, Rac 1 and Cdc42 as mediators in an acetyicholine (ACh)-activated signaling pathway that modulates OHC motility, offers now the opportunity to address this hypothesis at molecular and cellular levels. Moreover, our preliminary results indicate that some downstream effectors of Rho GTPases -belonging to the ACh-activated signaling pathway- are important for OHCs' mechanical homeostasis. Since these downstream effectors mediate Rho-promoted processes such as actin-spectrin association and plasma membrane-cytoskeleton interaction, these results provide a solid foundation for further investigation of the role of the cytoskeleton in the regulation of OHC motility. In this proposal, we will use a combination of electrophysiological, molecular biology, microscopical and image analysis techniques in order to: 1. Identify downstream effectors of Rho GTPases involved in the regulation of OHC motility, 2. Determine the connection pattern between Rho GTPases and their downstream effectors, and 3. Establish the structural changes induced by ACh on the OHC cytoskeleton. We are confident that accomplishing these aims will provide essential information about the role of the cytoskeleton in the regulation of OHC motility as well as critical insights into the basic mechanisms of both normal human hearing and deafness.